Has any tried utilizing UPLC-MS for the quantification of nitrites in raw materials?
I have seen papers using it to quantify the levels of nitrite in drinking water, with very low LOD and LOQ levels reported.
It would also remove the problems from chloride ions seen in standard IC analysis, and seems simpler than other techniques in terms of potential sample preparation, no derivatization required.
Or am I hoping for too much?
I think you are in the right track. The biggest problem with nitrite and nitrate analysis is the interference of chloride ions. As I used to do this for physiological fluids, I suffered a lot as I used HPLC and CEZ with 214 detection. But then I was not looking for such low levels as we are doing today. Body is full of nitrite and nitrate 
So, in my opinion, MS as a detector would possibly be the best option to get the chloride interference out of the way. Let us know your experience.
Nitrites as precursors of N-nitrosation in pharmaceutical samples – A trace level analysis - ScienceDirect
Perhaps this paper I recently shared in a different thread would be useful for you MarkS
Thank you @AntoniaW
This method looks to remove the need for any pre-derivatization in the sample prep.
[https://core.ac.uk/download/pdf/74395031.pdf]
But I don’t know if it would work in the presence of excipients, either individually, or if trying to ascertain a level in a complete placebo formulation.
I saw this one too and wondered the same. I will ask around and find out for you if there is any data for this I can find. Typically sub ppm levels were sufficient for excipient analysis but now I understand some are looking to try screen even lower.
@mayank.bhanti @Mrunal @AmandaGuiraldelli any insights or options explored on your side?
We do have a plan to evaluate orthogonal techniques such as the detection of derivatized nitrite and nitrate products using fluorescence, UV detectors, and mass detectors (SIM). We have not considered direct LC-MS (as reported in one of the publications on the detection of nitrite and nitrate in water) because it may be challenging to resolve different ions using reverse phase chromatography. The same was reported in the publication as well; however, they were still able to quantify different ions in SIM mode, probably because they were dealing with a water matrix. For excipients, it would be really hard to get rid of matrix interference chromatographically and matrix effect in MS ion source. We can still consider checking and determining if there would still be a way to resolve chromatographically and detect using MS.
@mayank.bhanti - thank you - I’m very far from a mass spec expert, hence my questions as I investigate all potential options.
It may be that the derivatization is the way to go from a mass spec perspective as well.
In my opinion IC (ion chromatography) can be a good choice for nitrite analysis, when using SPE for sample preparation.
Any update on this topic if we can use direct LCMS negative mode or the derivatized LCMS method?
@wamis8182 - I haven’t seen anything recently around using LCMS for the nitrite quantification.
I did find this just the other day - it is ion chromatography, but seems to be able to separate the nitrite from any chloride interference, and looks to work on entire pharmaceutical formulations, rather than just individual excipients.
I am currently working on publishing my nitrite method. It works with HPLC-UV (LOQ = 100 ppb) or HPLC-Fluorescence (LOQ = 10 ppb). Even difficult excipients or simply the finished products (tablets, gel, etc.) have shown good recoveries with my approach. This is a derivatization method, but the agent is relatively cheap and the method is quite easy to perform. I will keep you updated. Using LCMS was no option for us, since the instrument is always busy with nitrosamine-analysis.
this is very interesting Phil,
thanks for sharing it.
Looking forward for any final update,
best regards
Christos
Hi, Phil
What is the current status of this publication?
It is a work in progress and still takes time. I can link you the originating paper:
Determination of Nitrite and Nitrate in Cell Culture Medium by Reversed-Phase High-Performance Liquid Chromatography with Simultaneous UV-VIS and Fluorescence Detection | Jordan Journal of Chemistry (JJC)
Some small modifications were necessary but it is a really good starting point. Sample conc. used was 100mg/mL and LOQ was 1.0 ng/mL for us with Fluorescence detection. UV is about 10 times less sensitive if you don´t have the detector. As long as you keep the pH prior derivatization more or less neutral it worked for many excipients.
Regards,
Philipp
I have had some nice results with cations and anions using the Trinity P1 and P2 columns on CAD. The better nitrate-nitrite chromatography was found with the P2 using a 100 mM formate buffer and acetonitrile in a 40:60 ratio and UV analysis at 218 nm. The injection volume is critical with that column series due to their capacities. I couldn’t go over 1 uL with a 50 mm column which I believe is due to the triple phase combinations. Anything above that was causing more peak distortion than I would like.
As far as I know, Trinity P1 and P2 columns can be used to analyze cations and anions simultaneously. When it comes to nitrate and nitrite ions, I’m confused about whether CAD or UV has higher sensitivity in determining them.
My data showed the UV response @ 218 nm to be significantly stronger. On the order of about 25 times larger.
Can someone help? I was using an Acclaim Trinity P1 column with a UV detector to analyze the amount of nitrite in excipients. However, due to a problem with the column manufacturer, they are unable to ship columns that meet the specifications. I heard we won’t be getting the new columns until October. Are there any alternative columns that can effectively retain and separate nitrite and nitrate?
Thanks in advance,
Yosuke Mino
Hi, try using a Phenomenex Comixsil mixed-mode column—it effectively retains and separates nitrite and nitrate.